Pharmaceutical composition comprising substance inhibiting enzymatic activity of peroxiredoxin 2 as effective ingredient for treatment of colorectal cancer

ABSTRACT

The present invention relates to a pharmaceutical composition for treating colorectal cancer including a material inhibiting the enzyme activity of peroxiredoxin 2 as an active ingredient, and more specifically, to a pharmaceutical composition for treating colorectal cancer, which exhibits the effect of reducing colon polyps via increase of active β-catenin degradation by inhibiting the activity of peroxiredoxin 2, based on the mechanism that promotes colorectal tumor by the interaction between peroxiredoxin 2 (PrxII) and tankyrase (TNKS) in an APC-mutant cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation in Part of International ApplicationNo. PCT/KR2018/007316 filed Jun. 27, 2018, claiming priority based onKorean Patent Application No. 10-2017-0081491 filed Jun. 27, 2017 andKorean Patent Application No. 10-2018-0074375 filed Jun. 27, 2018, thecontents of all of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition fortreating colorectal cancer comprising a material inhibiting the enzymeactivity of peroxiredoxin 2 as an active ingredient.

BACKGROUND ART

Cancer is one of the most intractable diseases and is constantly beingstudied for the cure of cancer patients. In addition, in hospitals,techniques such as drug treatment, radiation therapy, gene therapy, etc.are being used to treat cancer.

Colorectal cancer (CRC) is a common cancer worldwide. In Korea,colorectal cancer is the most frequently occurring cancer in menfollowing gastric cancer, lung cancer, and liver cancer, and is the mostfrequently occurring cancer in women following breast cancer and gastriccancer. In recent years, as the diet of Koreans becomes westernized, theincidence has increased more rapidly. The mortality rate from colorectalcancer has increased by about 80% over the last 10 years and itcontinues to increase.

About 40-50% of CRC patients develop relapses and metastasis. The maincause of death in patients with malignant tumors, including CRC, is dueto the metastasis of a malignant tumor rather than the malignant tumoritself. Most cancer metastasis are multiple and systemic.

According to mass sequencing analysis data, more than 80% of CRCpatients have a mutation in the adenomatous polyposis coli (APC) gene.The APC protein is an important skeletal protein of the β-catenindestruction complex. Because of this, it is known that active β-cateninaccumulates Wnt-independently in cells with APC gene mutation and itinitiates the formation of intestinal tumor.

According to recent studies, the intestinal tumor formation triggered bythe APC gene mutation is promoted by the acquisition or inheritance of amutation in DNA glycosylase, which plays an important role in repair ofbases in nucleic acids at the time of oxidative DNA damage. This showsthat elevation in the levels of reactive oxygen species (ROS) isinvolved in the APC mutation-inducible intestinal tumor formation.

Peroxiredoxin (Prx) are known as peroxidases of hydrogen peroxide andalkyl hydroperoxide in vivo (Chae, H. Z. et al., Proc. Nat. Acad Sci.91: 7017-7021, 1994). Peroxiredoxin are classified as types I to VI ofPrx isozymes in mammals and are found in various parts of tissues (Rhee,S G et al., IUBMB Life 52: 35-41, 2001).

Peroxiredoxin (Prx) are known to exhibit a strong antioxidant activityin cells. Most of the peroxiredoxin isoenzymes, except for Prx type VI,use thioredoxin as an electron donor, and thus, they are known as athioredoxin peroxidase.

Prx family consists of six isozymes divided into 2-Cys (cysteine) and1-Cys (cysteine) subfamilies. The 2-Cys subfamily enzymes arethioredoxin-dependent peroxidases that are widely conserved frombacteria to humans. Among the 2-Cys Prx, cytoplasmic PrxI and PrxIIisozymes are known to be overexpressed in various types of cancer andplay an important regulatory role in membrane receptor-mediated signaltransduction.

Mammalian 2-Cys Prx enzymes receive electrons from an electron-conveyingsystem, which consists of thioredoxin-thioredoxin reductase, and reducehydrogen peroxide (H₂O₂) to water in the presence of nicotinamideadenine dinucleotide phosphate (NADPH).

Hydrogen peroxide regulates the reversible oxidation of signaltransduction proteins, including protein kinases and protein tyrosinephosphatase. Therefore, hydrogen peroxide functions as a potentialsecondary messenger in proliferating cancer cells. PrxII is known toregulate hydrogen peroxide locally. Therefore, PrxII, a 2-Cys Prxenzyme, is thought to play a multifaceted role in intracellular ROSdetoxification and signal transduction.

Tankyrase (hereinafter, TNKS) is a type of poly(ADP-ribose) polymerase.In CRC cells, the activity of tankyrase induces abnormal cellproliferation by inhibiting β-catenin degradation. Therefore, inhibitionof TNKS has been highlighted as a target for treating CRC, but there isa concern that direct inhibition of TNKS may cause pleiotropic effectsor side effects due to the presence of a wide range of substrates. Inaddition, the specific details of the mechanism that regulates theactivity of TNKS in CRC tumor formation are not yet known.

The axis inhibitor protein (Axin1), tumor suppressor, is anotherskeletal protein that constitutes the β-catenin destruction complex. Theregulatory mechanism of Axin1 protein by TNKS in APC-mutant cells hasnot been elucidated. In addition, the treatment of CRC, which regulatesthe activity of TNKS through the control of the redox system in vivo,has not been attempted until now.

SUMMARY OF THE PRESENT INVENTION

To solve the above-described problems in related art, an object of thepresent invention is to the mechanism on how peroxiredoxin 2 (PrxII)(which plays a multifaceted role in redox system and signal transductionin APC-mutant cells) regulates the activity of TNKS, and provides anovel pharmaceutical composition for treating CRC that can inhibit CRCby inhibiting the enzyme activity of PrxII by such a mechanism.

To solve the above-described problems in related art, in an embodimentof the present invention provides a pharmaceutical composition fortreating CRC comprising a material inhibiting the enzyme activity ofPrxII as an active ingredient.

In the pharmaceutical composition for treating CRC by the presentinvention, the material that inhibits the enzyme activity of PrxII isrepresented by the following Formula 1.

In Formula 1 above, R¹ is —O—R², a cyclic compound, or a compoundconsisting of H; and

R² is at least one selected from the group consisting of a C₁ to C₈, abranched or unbranched alkyl, alkenyl or alkynyl, and an aromatic ornon-aromatic cyclic compound which is substituted or unsubstituted.

In the pharmaceutical composition for treating CRC by the presentinvention, the material inhibiting the enzyme activity of PrxII mayinclude at least one compound selected from the group consisting ofCompound-1 to Compound-6 shown below.

In the pharmaceutical composition for treating CRC by the presentinvention, the material inhibiting the enzyme activity of PrxII ischaracterized in that it increases the degradation of β-catenin.

In the pharmaceutical composition for treating CRC by the presentinvention, the material inhibiting the enzyme activity of PrxII ischaracterized in that it decreases the degradation of Axin1 by TNKS.

In the pharmaceutical composition for treating CRC by the presentinvention, the material inhibiting the enzyme activity of PrxII ischaracterized in that it increases the oxidative inactivation of TNKS.

In the pharmaceutical composition for treating CRC by the presentinvention, the oxidative inactivation of TNKS occurs in the cytoplasm ofan APC-mutant cell.

In the pharmaceutical composition for treating CRC by the presentinvention, the material inhibiting the enzyme activity of PrxII ischaracterized in that it decreases the interaction between PrxII andTNKS.

The pharmaceutical composition for treating CRC by the present inventionis characterized in that it contains the material inhibiting the enzymeactivity of PrxI in a pharmaceutically effective amount. As used herein,the term “pharmaceutically effective amount” refers to an amount whichis sufficient to achieve the efficacy or activity of the materialinhibiting the enzyme activity of PrxII.

The pharmaceutically acceptable carriers that can be included in thepharmaceutical composition are those commonly used in the preparation offormulations, and these pharmaceutically acceptable carriers may includelactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber,calcium phosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc,magnesium stearate, mineral oil, etc. but are not limited thereto.

The pharmaceutical composition may further include lubricants, wettingagents, sweeteners, flavoring agents, emulsifiers, suspending agents,preservatives, etc. in addition to the above components. Suitablepharmaceutically acceptable carriers and formulations are described indetail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition may be prepared in unit-dose form orformulated through incorporation into a multi-dose container byformulating using a pharmaceutically acceptable carrier and/orexcipient, in accordance with methods readily available to one ofordinary skill in the art to which this invention pertains. Inparticular, the formulations may be in the form of solutions,suspensions, syrups, or emulsions in oils or aqueous media, or in theform of extracts, powders, granules, tablets or capsules, and mayfurther include dispersants or stabilizers.

The features, forms, and advantages of one embodiment of the presentinvention are set forth in the description below, and in part can beapparent from the description or can be learned by practice of suchexemplary embodiments.

Other features, forms, and advantages will be apparent to those skilledin the art from the following description and claims, or may be learnedby practicing the embodiments described below.

ADVANTAGEOUS EFFECTS

According to an embodiment of the present invention, a composition,which can reduce colorectal polyps via regulation the redox system ofCRC cells by inhibiting the enzyme activity of PrxII and can treat orprevent CRC, can be provided. According to an embodiment of the presentinvention, a pharmaceutical composition, which can reduce colorectalpolyps by reducing the interaction between PrxII and TNKS in thecytoplasm of an APC-mutant cell and can treat or prevent CRC, can beprovided. According to an embodiment of the present invention, a methodfor treating CRC which can inhibit the activity of TNKS by regulation ofthe intracellular redox system can be provided.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1 and 2 show images illustrating the results of confirming thegenotype of a double-mutant mouse prepared according to an embodiment ofthe present invention.

FIG. 3 shows images illustrating the results of PrxII protein expressionby immunofluorescence staining in non-polyp segments and polyps of thesmall intestine according to an embodiment of the present invention.

FIG. 4 shows microscopic images illustrating the intestinal tissue of adouble-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 5 shows graphs illustrating the number of polyps in the intestinaltissue of a double-mutant mouse prepared according to an embodiment ofthe present invention.

FIG. 6 shows images illustrating the results of staining thecross-sections of the small intestine and colon of a double-mutant mouseprepared according to an embodiment of the present invention withhematoxylin and eosin.

FIG. 7 shows microscopic images illustrating the intestinal tissue of adouble-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 8 shows graphs illustrating the number of polyps in the intestinaltissue of a double-mutant mouse prepared according to an embodiment ofthe present invention.

FIG. 9 shows images illustrating the results of immunoblotting performedusing an intestinal tissue extract of a double-mutant mouse preparedaccording to an embodiment of the present invention. Graphs showquantified data of immune-reactive bands.

FIG. 10 shows graphs illustrating the results of the expression levelsof Axin1, (3-catenin, and β-catenin target genes in the polyp tissue ofa double-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 11 shows images and graphs illustrating the results ofimmunohistochemistry image analysis for Ki-67 in the polyp tissue of adouble-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 12 shows images and a graph illustrating the results of theproliferating cells analysis by the fluorescence staining, withanti-bromo uridine (anti-BrdU) antibody in the polyp tissue of adouble-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 13 shows images and graphs illustrating the results of theproliferating cell analysis by the fluorescence staining, withanti-bromo uridine (anti-BrdU) antibody in the polyp tissue of adouble-mutant mouse prepared according to an embodiment of the presentinvention.

FIG. 14 shows images and a graph illustrating the results of tunnel(TUNEL) staining of the apoptosis in the polyp tissue of a double-mutantmouse prepared according to an embodiment of the present invention.

FIG. 15 shows images illustrating the results, in which cells weretransfected with a set of siRNAs specific for PrxI and PrxII for 48hours and immunoblotted against β-catenin, according to an embodiment ofthe present invention.

FIG. 16 shows images illustrating the results, in which cells weretransfected with PrxII-1 siRNA at various concentrations for 48 hoursand immunoblotted against β-catenin, according to an embodiment of thepresent invention.

FIG. 17 shows images illustrating the results, in which variousAPC-mutant colorectal cancer cell (CRC) lines were transfected withPrxII-1 siRNA for 48 hours and immunoblotted against β-catenin andactive β-catenin, according to an embodiment of the present invention.

FIGS. 18 and 19 show images illustrating the results, in which variousAPC-mutant colorectal cancer cell (CRC) lines were transfected withPrxII-1 siRNA for 48 hours, infected again with retroviruses expressingthe active (WT) or inactive (CS or C172S) forms of PrxII, andimmunoblotted against β-catenin and active β-catenin, according to anembodiment of the present invention.

FIG. 20 shows graphs illustrating the results of the PrxII-dependentH₂O₂ levels in APC-mutant CRC cells, according to an embodiment of thepresent invention.

FIGS. 21 to 23 show images confirming that β-catenin destruction complexis involved in CRC, in which a control group according to an embodimentof the present invention and PrxII-deficient colorectal cancer cell(CRC) lines (i.e., SW480 and HT29) were each treated with a proteasomeand a glycogen synthase kinase-β (GSK3β) inhibitor (25 μM MG132,lactacystin, 2.5 μM BIO, and SB216763) for one hour, and the expressionlevels of β-catenin and active β-catenin were measured.

FIG. 24 shows an image confirming that PrxII deficiency does not affectthe activation of GSK3β in APC mutant colorectal cancer (CRC) cellsaccording to an embodiment of the present invention.

FIG. 25 shows an image confirming that PrxII deficiency does not affectnormal Wnt3A signal stimulation in HEK293 cell line according to anembodiment of the present invention.

FIG. 26 shows a graph illustrating the results of β-catenin/TCFtranscription activity according to an embodiment of the presentinvention.

FIGS. 27 to 32 each show an image illustrating the analysis results ofgenes expressed at different levels in HT29 and SW480 cells due to PrxIIdeficiency according to an embodiment of the present invention.

FIG. 33 shows an image illustrating the results of transfection of HT29and SW480 cells with a control or PrxII-1 siRNA followed by measurementby immunoblotting against active β-catenin and Axin1, according to anembodiment of the present invention.

FIG. 34 shows an image confirming that PrxII deficiency significantlyincreases complexes, in which SW480 cells according to an embodiment ofthe present invention were transfected with a control or PrxII-1 siRNAand immunoblotting was performed for all complex-binding proteinsseparated by immunoprecipitation (IP) of Axin-1.

FIG. 35 shows an image illustrating the results of transfection of HT29cells with a control or Axin-1/Axin-2 siRNAs, followed by immunoblottingagainst active i-catenin and Axin1, according to an embodiment of thepresent invention.

FIG. 36 shows images illustrating the results of transfection of HT29and SW480 cells with a control or PrxII-1 siRNA, and immunoprecipitationof Axin-1, followed by immunoblotting performed on ubiquitination andpoly-ADP-ribosylation, according to an embodiment of the presentinvention.

FIG. 37 shows an image and a graph illustrating the results of a colonyforming assay, which was performed after transfecting RKO (i.e., an APCwild-type colorectal cancer (CRC) line) and HT29 and SW480 cells (i.e.,APC-mutant cell lines) with a control or PrxII-1 siRNA, according to anembodiment of the present invention.

FIG. 38 shows an image and graphs illustrating the results of a colonyforming assay, which was performed after transfecting HT29 and SW480cells (i.e., APC-mutant cell lines) with a control or PrxII-1 siRNA,followed by expression of activated β-catenin (S37A), according to anembodiment of the present invention.

FIGS. 39 and 40 each show images illustrating the results, in which HT29and RKO cells were transfected with a control or PrxII-1 siRNA, ortreated with H₂O₂, and TNKS1 was immunoprecipitated, and the activity ofADP-ribose polymerase (PARP) was measured, according to an embodiment ofthe present invention.

FIG. 41 shows images illustrating the results, in which various APCwild-type and APC-mutant colorectal cancer cell (CRC) lines weretransfected with a control or PrxII-1 siRNA, and the expression levelsof TNKS1, and Axin1 and TRF1 (i.e., the substrates of TNKS1) weremeasured by immunoblotting, according to an embodiment of the presentinvention.

FIG. 42 shows images illustrating the results, in which various APCwild-type and APC-mutant colorectal cancer cell (CRC) lines werepretreated with a TNKS1 inhibitor (XAV939) for one hour, and theexpression levels of TNKS1, and Axin1 and TRF1 (i.e., the substrates ofTNKS1) were measured by immunoblotting, according to an embodiment ofthe present invention.

FIG. 43 shows graphs illustrating the results, in which variousAPC-mutant colorectal cancer cell (CRC) lines were pretreated with aTNKS1 inhibitor (XAV939) for one hour, and the proliferation levels ofthe cells were measured, according to an embodiment of the presentinvention.

FIG. 44 shows a graph illustrating the results of the intracellular H₂O₂levels in APC gene deficient RKO colorectal cancer (CRC) cell lines,according to an embodiment of the present invention.

FIG. 45 shows an image illustrating the results, in which the levels ofβ-catenin, Axin1, and TNKS1 were measured by immunoblotting in APC genedeficient RKO cells, according to an embodiment of the presentinvention.

FIG. 46 shows images illustrating the results of ADP-ribose polymeraseactivity of TNKS1 in APC gene deficient RKO cells, according to anembodiment of the present invention.

FIG. 47 shows images illustrating the results, in which the cellsexpressed various TNKS1 cysteine single mutant enzymesimmunoprecipitated with an anti-TNKS antibody, and the ADP-ribosepolymerase activity of TNKS1 was measured, according to an embodiment ofthe present invention.

FIG. 48 shows an image illustrating the results of expression andpurification of recombinant proteins in E. coli cells for the PARPdomain of TNKS1 according to an embodiment of the present invention.

FIG. 49 shows a graph illustrating the results of the releasing of Zincions by hydrogen peroxide treatment in the purified recombinantTNKS1-PARD protein according to an embodiment of the present invention.

FIG. 50 shows images illustrating the results of immunoblotting of thecells against the proteins indicated after the immunoprecipitationreaction with an anti-TNKS antibody, according to an embodiment of thepresent invention.

FIG. 51 shows images illustrating the results of Co-immunoprecipitation(co-IP) for detecting a binding between TNKS and PrxII proteins,according to an embodiment of the present invention.

FIGS. 52 and 53 each show images illustrating the results of in situproximity ligation assays according to an embodiment of the presentinvention.

FIGS. 54 and 55 each show images illustrating the results of mutualbinding assays using a truncation mutation or a single point mutation ofTNKS and Myc-PrxII, respectively, according to an embodiment of thepresent invention.

FIGS. 56 and 57 illustrate the results of intracellular expression andperoxidase activity of wild type and a G116V mutant of PrxII accordingto an embodiment of the present invention.

FIG. 58 shows images and a graph illustrating the results of PARPactivity of TNKS1 in cells expressing wild type and a G116V mutant ofPrxII according to an embodiment of the present invention.

FIG. 59 shows an image and a graph illustrating the results of colonyforming assays in cells expressing wild type and a GI 16V mutant ofPrxII according to an embodiment of the present invention.

FIGS. 60 and 61 each show images illustrating the comparison results ofexpression levels of PrxI and PrxII genes in CRC patients (n=155)retrieved from healthy individuals and The Cancer Genome Atlas (TCGA)code database according to an embodiment of the present invention.

FIG. 62 shows images and a graph illustrating the results of PrxIIimmunostaining in colon tissue arrangement of healthy individuals andCRC patients according to an embodiment of the present invention.

FIG. 63 shows a graph illustrating the results of inhibition of activityagainst PrxI and PrxII of Compound-6 (Conoidin A) according to anembodiment of the present invention.

FIG. 64 shows an image and a graph illustrating the results of colonyforming assays in colorectal cancer (CRC) cell lines pretreated withCompound-6 (Conoidin A) according to an embodiment of the presentinvention.

FIGS. 65 and 66 each show the results of luminescence imaging in vivoand weight measurement of tumor growth after transplantation(xenograft), to a mouse, of the colorectal cancer (CRC) cell line, HT29,treated with Compound-6 (Conoidin A) according to an embodiment of thepresent invention.

FIG. 67 shows a schematic diagram illustrating the effects of inhibitingCRC by the suppression of the enzyme activity of PrxII according to anembodiment of the present invention.

FIG. 68 shows an image illustrating the results of the expression levelsof PrxII gene in CRC patients with APC WT or an APC mutation accordingto an embodiment of the present invention.

FIG. 69 shows a graph illustrating the results of peroxidase activitymeasured after reacting PrxII with Compound-1 in vitro according to anembodiment of the present invention.

FIG. 70 shows a graph illustrating the results of peroxidase activitymeasured after reacting PrxII with Compound-2 in vitro according to anembodiment of the present invention.

FIG. 71 shows a graph illustrating the results of peroxidase activitymeasured after reacting PrxII with Compound-3 in vitro according to anembodiment of the present invention.

FIG. 72 shows a graph illustrating the results of peroxidase activitymeasured after reacting PrxII with Compound-4 in vitro according to anembodiment of the present invention.

FIG. 73 shows a graph illustrating the results of peroxidase activitymeasured after reacting PrxII with Compound-5 in vitro according to anembodiment of the present invention.

FIGS. 74A and 74B show the results of RKO cell colony cultureexperiments in Conoidin A and Compound-1 to Compound-5 according to anembodiment of the present invention.

FIGS. 75A and 75B show the results of HT29 cell colony cultureexperiments in Conoidin A and Compound-1 to Compound-5 according to anembodiment of the present invention.

MODE FOR CARRYING OUT THE INVENTION

The advantages and features of the present invention, and a method ofachieving the same will be apparent with reference to the followingExamples to be described hereinbelow in conjunction with theaccompanying drawings. However, these Examples are intended toillustrate the present invention in more detail, and the scope of thepresent invention is not limited by the following Examples.

Throughout the specification, when a part includes a certain component,this means that it may further include other components rather thanexcluding other components, unless otherwise specified.

As used herein, the terms Compound-1 to Compound-6 refer to thematerials shown in Table 1 below.

Compound-1 2,3-bis(bromomethyl)-6- methoxyquinoxaline 1,4-dioxide

Compound-2 2,3-bis(bromomethyl)-6- ethoxyquinoxaline 1,4-dioxide

Compound-3 2,3-bis(bromomethyl)-6- isopropoxyquinoxaline 1,4- dioxide

Compound-4 6-(alloyloxy)-2,3- bis(bromomethyl)quinoxaline 1,4-dioxide

Compound-5 2,3-bis(bromomethyl)-6-(prop-2- ynyloxy)quinoxaline1,4-dioxide

Compound-6 2,3-bis(bromomethyl)quinoxaline 1,4-dioxide

<Example 1> Cell Culture

All CRC and HEK293 cells were provided by the American Type CultureCollection (Manassas, Va., USA). SW480, DLD1, CoLo205, Colo741, andSW620 cells were subcultured in RPMI 1840 medium supplemented with 10%fetal bovine serum.

HEK293 and RKO cells were cultured in Dulbecco's Modified Eagle's Mediumwith 10% FBS. HT29 cells were cultured in McCoy's 5A medium with 10%FBS. Mycoplasma contamination was periodically tested in cell culturesupernatants using the mycoplasma detection kit (Biotool, USA).

<Example 2> Preparation of Double-Mutant Mice

To test the CRC-specific function of PrxII in wivo, double-mutant micewere prepared by crossing PrxI^(+/−) and PrxII^(+/−) mice withAPC^(Min/+) mice.

PrxI^(+/−) and PrxII^(+/−) C57BL/6 mice were crossed with APC^(Min/+)mice according to the C57B/6 background (Jackson Laboratory, Bar Harbor,USA), bred and maintained in aseptic facilities, and thereby complexmutants representing the genotypes ofAPC^(Min/+);PrxI^(−/−),APC^(Min/+);PrxI^(+/−), APC^(Min/+);PrxI^(−/−),APC^(Min/+);PrxII^(+/−), APC^(Min/+);PrxII^(−/−), andAPC^(Min/+);PrxII^(−/−) were prepared.

The genotypes of the littermates were confirmed by performing genomicPCR for mouse tail DNA using specific primers therefor. The littermatesrefers to babies born from the same mother.

All mouse experiments were approved by the Institutional Animal Care andUse Committee (IACUC) of Ewha Womans University and performed incompliance with the ARRIVE guidelines. Animal experiments were performedby a double-blind test after separation of animal breeding and tissueanalysis.

In the case of a tumor xenograft model, the mice were anesthetized bythe inhalation of isoflurane gas (N₂O:O₂/70%:30%) and subcutaneouslyinjected with HT29-luc2 cells (2.5×10⁵ cells) suspended in 200 μL ofPBS. Compound 1 to Compound 6 (286 μM in DMSO) were intraperitonealadministered starting from 6 days after the cell injection and repeatedevery 3 days.

Bioluminescent imaging was performed with IVIS Lumina Series III (PerkinElmer). For each image photographing session, luciferin suspended in PBS(150 mg of luciferin/kg body weight) was administered intraperitoneallyaccording to the manufacturer's protocol.

Up to four animals were maintained in the integral anesthetic manifoldequipment and they were imaged 10 minutes after luciferin injection. TheIVIS imaging system collects the photographic images of mice andquantitative bioluminescent signals therefrom and then allows them tooverlap with each another.

<Example 3> Genotyping of Double-Mutant Mice

The genotypes of the mice, to which a double mutation was induced,prepared in Examples above were determined by performing genomic PCR atWeek 4, and the resulting genotypes of the double-mutant mice preparedin Examples above are shown in FIGS. 1 and 2.

In FIGS. 1 to 3, it confirmed that the double mutant mice prepared inExamples above can develop multiple intestinal neoplasia (Min) by atruncation mutation of adenomatous polyposis coli (APC). The products ofthe truncated APC gene in FIGS. 1 and 2 are indicated by asterisks.

Although APC mutations are heterozygous, intestinal adenomatouspolyposis is known to be caused by loss of residual APC wild type (WT),and the resulting adenomatous polyposis is known to include a truncatedAPC wild type (WT) copy similar to that of human CRC tumor.

The primers used to determine genotypes are as follows.

APC^(Min) (wild type) (SEQ ID NO: 1) 5′-GCCATCCCTTCACGTTAG-3′ (mutant)(SEQ ID NO: 2) 5′-TTCTGAGAAAGACAGAAGTTA-3′ (common) (SEQ ID NO: 3)5′-TTCCACTTTGGCATAAGGC-3′ PrxI forward (SEQ ID NO. 4)5′-CTGGAAACCTGGCAGTGATA-3′ reverse, (SEQ ID NO: 5)5′-CTGTGACTGATAGAAGATTGGT-3′ PrxII forward, (SEQ ID NO. 6)5′-GATGATCTCCGTGGGGCAAACAAA-3′ reverse, (SEQ ID NO: 7)5′-ATGGCCTCCGGCAACGCGCAAATC-3′ Neo cassette forward, (SEQ ID NO. 8)5′-GCTTGGGTGGAGAGGCTATTCG-3′ reverse,  (SEQ ID NO: 9)5′-GTAAAGCACGAGGAAGCGGTCAGC-3′

<Example 4> Observation of Adenomatous Polyps in Double-Mutant Mice

The small intestine and colon were excised from the mouse, theadenomatous polyps of the intestine were separated and observed using astereomicroscope, and the results are shown in FIG. 4.

The number of polyps whose diameter exceeds 0.3 mm in the smallintestine and colon of the mice was measured and the results are shownin FIG. 5. As shown in FIG. 5, the average number of polyps greater than0.3 mm in diameter shown in the small intestine and colon of theAPCN^(Min/+);PrxII^(−/−) mouse was decreased by about 50% compared tothose shown in the APC^(Min/+);PrxII^(−/−) mouse and theAPC^(Min/+);PrxII^(+/−) mouse.

The APC^(Min/+);PrxII^(−/−) mouse (average days of survival=241 days)survived much longer than the APC^(Min/+);PrxII^(+/+) mouse (averagedays of survival=146 days) and the APC^(Min/+);PrxII^(+/+) mouse(average days of survival=152 days).

Histological examination of the small and large intestines of the micewas performed and the results are shown in FIG. 6. As shown in FIG. 6,PrxII deficiency did not change the villi structure but decreased thefrequency and size of the adenomatous polyps.

The intestinal adenomatous polyps of PrxI-deficient mice were observedusing a stereomicroscope and the numbers thereof were counted and areshown in FIGS. 7 and 8. The average number of intestinal polyps of theAPC^(Min/+);PrxI^(−/−) mouse was the same as those ofAPC^(Min/+);PrxI^(+/+) mouse and the APC^(Min/+);PrxI^(+/−) mouse.

From the above results, it confirmed that while PrxII promotesintestinal tumor formation induced by an APC mutation in vivo, PrxI isindependent of intestinal tumor induced by an APC mutation.

<Example 5> Measurement of β-Catenin Expression

The expression levels of β-catenin and its gene in the polyps separatedfrom the APC^(Min/+);PrxII^(+/+) mouse and the APC^(Min/+);PrxII^(−/−)mouse were measured by immunoblotting and the results are shown in FIG.9.

As shown in FIG. 9, the expression levels of β-catenin and itstranscription targets (i.e., c-Myc and cyclin D1) in theAPC^(Min/+);PrxII^(−/−) tumor were significantly reduced compared tothose in the APC^(Min/+);PrxII^(+/+) tumor. However, the expressionlevel of Axin1, an important scaffold protein in the β-catenindestruction complex, increased in APC^(Min/+);PrxII^(−/−) mouse ininverse proportion to tumors.

As shown in FIG. 10, since the expression levels of β-catenin and Axin1mRNA did not change between APC^(Min/+);PrxII^(+/+) andAPC^(Min/+);PrxII^(−/−), it confirmed that PrxII regulates theexpression of Axin1 and β-catenin at the protein level in vivo.

<Example 6> Determination on Whether β-Catenin Target Genes are Involvedin Proliferation and Survival of CRC Cells

The number of proliferating cell and dead cells were counted todetermine whether β-catenin target genes are involved in theproliferation and survival of CRC cells.

As shown in Ki-67 expression and BrdU incorporation assays in FIGS. 11and 12, the proportion of proliferating cells was similar in the polypsof APC^(Min/+);PrxII^(+/+) and APC^(Min/+);PrxII^(−/−).

In addition, in FIG. 13, the BrdU incorporation assays showed that PrxIIdeficiency had no effect at all on the proliferation and migration ofintestinal epithelial cells of the scrotum.

In contrast, in FIG. 14, the number of dead cells measured by TUNELstaining in the polyp of the APC^(Min/+);PrxII^(−/−) was significantlyhigher than the number of dead cells measured by TUNEL staining in thepolyp of the APC^(Min/+);PrxII^(+/+). These results suggest that PrxIIpromotes survival of tumor cells in intestinal adenomatous polypsinduced by APC mutations.

<Example 7> Analysis of Regulation Mechanism β-Catenin Expression byPrxII

The mechanism of regulating β-catenin expression by PrxII in human CRCcells overexpressing PrxII was analyzed.

As shown in FIG. 15, the siRNA analysis in both APC-mutant CRC cells(i.e., SW480 and HT29 cells) showed that the decrease of PrxIIexpression resulted in a significant decrease in the expression levelsof endogenous β-catenin and expression of a truncated mutant form of APCprotein. However, PrxI deficiency did not change the β-cateninexpression in both SW480 and HT29 cells.

In addition, as shown in FIG. 16, the decrease of R-catenin expressionis proportional to the degree of PrxII deficiency, and from this result,it was indicated that a strict knockdown of PrxII is important forreducing β-catenin expression level.

As shown in FIG. 17, PrxII deficiency also reduced the expression levelsof total β-catenin and active β-catenin (non-phosphorylated form) inother APC-mutant CRC cells (i.e., SW620, DLD-1, and CoLo205).

To exclude off-target effects of PrxII siRNA, PrxI was expressed bytransfecting HT29 cells with PrxII, which is in an siRNA-resistant form.

As shown in FIGS. 18 and 19, it confirmed that PrxII wild type (WT)plays a specific role of PrxII that regulates β-catenin expression byfully restoring total β-catenin and active β-catenin expression levels,compared to siRNA-transfected control cells.

In contrast, it confirmed that the expression of PrxII inperoxidase-inactive mutants (i.e., C172S and C51/172S) cannot restoreβ-catenin expression levels, thus suggesting that peroxidase activity ofPrxII is necessary to maintain active β-catenin levels in CRC cells. InFIG. 20, it confirmed that the PrxII deficiency increases the amount ofintracellular hydrogen peroxide levels in HT29 and SW480 cells.

Since it was confirmed that PrxII deficiency did not change β-cateninmRNA levels, the degradation of β-catenin protein by a canonicaldestruction complex, which shows a sequential phosphorylation ofβ-catenin by casein kinase−1 and glycogen synthase kinase-3β (GSK-3β),was measured.

Since phosphorylated β-catenin is known to be ubiquitonated by anenzyme, which is called β-transducin repeats-containing proteins(β-TrCP) or ubiquitin E3 ligase, and degraded by a porotesome, it wastested whether the β-catenin canonical destruction complex is involvedin PrxII-dependent regulation of β-catenin expression.

In FIGS. 21 to 23, the GSK-3β inhibitor increased the activity ofβ-catenin, which indicates that constitutively-active GSK3β is involvedtherein.

However, as shown in FIG. 24, it was indicated from the tyrosinephosphorylation level of GSK3β, which is an indicator of kinaseactivation, that PrxII deficiency is unlikely to stimulate GSK3βactivation.

In addition, in FIG. 25, it was observed that PrxII deficiency does notaffect Wnt-induced β-catenin stabilization, and it confirmed that PrxIIselectively participates in deregulated β-catenin signaling.

In FIG. 26, as a result of decreased β-catenin expression, PrxIIdeficiency induced a significant decrease in TCF-dependent reporterexpression.

<Example 8>β-Catenin/TCF-Dependent Regulation of Transcription by PrIIDeficiency

The β-catenin/TCF-dependent transcription in HT29 cells was examined bymRNA sequencing via mRNA sequence analysis and the results are shown inFIGS. 27, 28, 29, and 30.

As shown in FIG. 31, in particular, PrxII deficiency downregulated 12 ofthe β-catenin target genes, which are expressed in CRC cells thatinclude major β-catenin target genes (e.g., CCND1, AXIN2, and BIRC5)(FDR<0.05).

Other metastatic and cell cycle promoting genes (e.g., S100A4, MMP7,ID2, and PTTG1) were also downregulated by PrxII deficiency.

In addition to HT29 cells, PrxII deficiency downregulated 13 β-catenintarget genes in SW480 cells (FDR<0.05), and in FIG. 32, it indicatedthat four genes (i.e., CCND1, S100A4, ID2, and EDN1) overlap with oneother.

The downregulation of several different genes in SW480 cells indicatesthat it may mediated by the secondary effect of decrease in theβ-catenin-related transcription complex or β-catenin.

These results indicate that PrxII deficiency promotes the degradation oftranscription-active β-catenin through the destruction complex of CRCcells.

<Example 9> Wnt-Independent and Axin1-Dependent Destruction of β-Cateninby PrxII Deficiency in CRC Cells

Axin1, another scaffold protein in APC mutations, is known to play animportant role in β-catenin destruction. The overexpression of Axin1 inAPC-mutant CRC cells is known to sufficiently induce the degradation ofβ-catenin.

Previously in FIG. 9, it confirmed that the expression of Axin1 wasincreased in the intestinal polyps of mice in which the PrxII gene wasremoved.

The amount of Axin and the Axin1-related destruction complex weremeasured in CRC cells. As a result of the immunoblot analysis in FIG.33, it confirmed that PrxII deficiency increased the expression ofendogenous Axin1 protein in both HT29 and SW480 CRC cells, whichindicates that PrxII deficiency is inversely correlated with theexpression of active β-catenin.

From the co-immunoprecipitation experiment in FIG. 34, it indicated thatthe PrxII deficiency increased the expression of the Axin1-relateddestruction complex. The phospho-β-catenin disappeared from the complexupon treatment with BIO, which is a GSK3β inhibitor.

In addition, in FIG. 35, the knockdown of Axin1/2 restored the activeβ-catenin levels of PrxII-deficient cells to the levels of cells in thecontrol.

These results indicate that Axin forms a functional destruction complexand regulates β-catenin degradation in PrxII-deficient CRC cells.

Since Axin is degraded by poly(ADP-ribose) polymerization (PARsylation)and subsequent ubiquitination, the state of Axin1 in HT29 and SW480cells was analyzed.

In fact, it confirmed that the treatment with MG132 (i.e., a proteasomeinhibitor) induces the accumulation of PARsylated and ubiquitinatedAxin1 in the cells of the control and thus, Axin1 continues to degrade.

In contrast, as shown in FIG. 36, PrxII deficiency inhibited thePARsylation/ubiquitination of Axin1 without affecting totalintracellular ubiquitination.

<Example 10> Determination of Colony Forming Ability of PrxII-DeficientCRC

To evaluate the biological significance of the PrxII-dependentregulatory mechanism of the Axin1/β-catenin pathway, colony formingability in CRC cells was examined.

As a result of the in vitro colony forming ability assay, in FIG. 37, itconfirmed that PrxII deficiency sufficiently inhibits colony formationin HT29 and SW480 cells, not RKO cells that express APC WT.

In FIG. 38, it confirmed that the expression of the active β-cateninS37A mutant almost completely restores the colony forming ability ofAPC-mutant CRC cells damaged by PrxII deficiency.

From these results, it indicated that PrxII deficiency can sufficientlyreverse the carcinogenic phenotype of APC mutants via induction ofβ-catenin degradation by Axin1.

<Example 11> Analysis of Regulation of TNKS-Axin1 Signaling System byPrxII Deficiency

TNKS is the only enzyme for poly(ADP-ribose) polymerization(PARsylation) of Axin proteins. Whether PrxII is essential for TNKSactivity was confirmed by performing in vitro PARP analysis.

As shown in FIG. 39, PrxII deficiency induced a severe impairment ofTNKS activity in APC-mutants (i.e., HT29 and SW480 cells), but not inRKO cells in which the AKO-function is retained. As shown in FIG. 40, onthe contrary, hydrogen peroxide treatment inhibited TNKS activity inboth HT29 and SW480 cells.

From these results, it may indicated that exogenous hydrogen peroxidedirectly inactivates TNKS activity regardless of APC mutation.

Since TNKS is known to be subjected to auto-polyADP-ribosylation (autoPARsylation) and degradation, the expression level of TNKS was measuredin the CRC cell panel along with the substrate proteins.

As shown in FIG. 41, PrxII deficiency increased the expression levels ofTNKS and Axin1 in all APC-mutant CRC cells tested, but not in cellsincluding RKO and Colo741 cells where APC-function was retained.

More importantly, the PrxII deficiency did not affect the expressionlevel of telomeric repeat-binding factor 1 (TRF1), which is another TNKSsubstrate essential for telomerase regulation in the nucleus.

In FIGS. 42 and 43, on the contrary, direct inhibition of TNKS using aspecific inhibitor, XAV939, increased the expression levels of TNKS andthe substrates (i.e., Axin1 and TRF1) in all CRC cells, resulting ininhibition of proliferation of HT29 and SW480 cells.

APC knockdown was performed in RKO cells to confirm a direct correlationbetween APC and PrxII function in CRC cells.

As shown in FIGS. 44, 45, and 46, the APC knockdown certainly induced asignificant increase in the expression levels of H₂O₂ and β-catenin incells, and consequently accelerated H₂O₂-dependent inactivation ofTNKS1. In addition, simultaneous deficiency of APC and PrxII increasedthe expression levels of TNKS and Axin1 proteins along with a decreasein the expression level of β-catenin.

Since PrxII is a peroxidase in the cytoplasm, it is suggested that PrxIIcan selectively protect TNKS in the cytoplasm from oxidative stressinduced by APC mutation or loss.

<Example 12> Confirmation of Mechanism of Inactivation of PARP Activityof H₂O₂-Mediated TNKS

To confirm the mechanism of inactivation of PARP activity ofH₂O₂-mediated TNKS, the oxidation-sensitive Cys residues within the PARPcatalytic domain of TNKS were examined.

Five Cys residues including three Zn-binding motifs were found byaligning the peptide sequences of various PARP domains, and these Cysresidues were found to be uniquely present in the TNKS isomers betweenPARP family.

In TNKS1, each Cys residue was mutated to Ser and its PARP activity wasexamined. As a result, the mutations of three Zn-coordinating Cysresidues (C1234, C1242, and C1245) among the five Cys residues foundresulted in a complete loss of PARP activity, as shown in FIG. 47.

To test whether zinc binding motifs are unstable under oxidizingconditions, a recombinant TNKS1 PARP domain (amino acids of 1023-1327)was prepared. The TNKS1 PARP domain showed intact polyADP-ribosylationactivity and was fully inactivated by incubation with H₂O₂, as shown inFIG. 48.

Zinc binding proteins release zinc ions by cysteine oxidation, and thereleased free zinc ions can be measured by a spectrophotometer using4-(2-pyridylazo) resorcinol.

The results measured by a spectrophotometer by the above method areshown in FIG. 49. In FIG. 49, it indicated that H₂O₂ treatment induces anearly complete release of zinc ions such that 90% or more of TNKS1 PARPproteins ultimately lost zinc ions.

From these results, it confirmed that zinc binding motifs of TNKS areessential for PARP activity and that the oxidation of Cys residuesmediated by H₂O₂ can induce the release of zinc ions from the PARPdomain.

From these results, it indicated that the redox of TANKS is regulated byPrxII-regulated H₂O₂.

<Example 13> Examination of Selective Binding Between PrxII and TNKS

The interaction between PrxII and TNKS was examined to demonstrate howPrxII protects TNKS from the H₂O₂-mediated inactivation.

In FIG. 50 which shows the experimental results ofco-immunoprecipitation (co-IP), it confirmed that endogenous TNKSinteracts with PrxII only in APC-mutant HT29 and SW480 cells, but not inRKO cells where APC functions are retained.

In contrast. TNKS did not interact with PrxI in APC-mutant HT29 andSW480 cells, confirming the specific role of PrxII in theTNKS/Axin1/β-catenin pathway.

To characterize the direct interaction between TNKS1 and PrxII, twoproteins were overexpressed in human embryonic kidney cells, HEK293, asnon-CRC cells. As a result, as shown in FIG. 51, it confirmed that TNKSand PrxII directly interact in the co-IP experiment.

More specifically, in FIGS. 52 and 53, the in situ proximity ligationassay (in situ PLA) visualized that the direct interaction between TNKSand PrxII occurs in the cytoplasm of HT29 and SW480 cells, not in RKOcells. In FIGS. 52 and 53, the red fluorescence signals indicating theinteraction of the two proteins disappeared almost completely by PrxIIdeficiency.

Therefore, it indicated that the blocking of redox in TNKS by PrxII ishighly specific of a protein-protein interaction and is dependent on APCmutations.

TNKS and PrxII mutagenesis were performed to analyze molecularinteraction maps. In FIG. 54, IP experiments showed that when atruncated TNKS mutant is expressed along with PrxII, PrxII interactswith the ankyrin repeat cluster (ARC) 4/5 domain of TNKS. Thisdemonstrated that PrxII binding does not overlap with Axin1, which bindsto the ARC 2/3 domain.

The TNKS ARC domain recognizes the consensus sequence, RXXPXG(SEQ ID NO:10), in the client protein, and in particular, the Gly residue atposition 6 plays a crucial role in direct binding.

After searching similar hexapeptide sequences in PrxII, three potentialcommon regions were found, and Gly-to-Val mutations were introducedthereto. In FIG. 55 in which the results of the Co-IP experiment areshown, it indicated that only the G116V mutation among the threemutation sites can completely remove the binding between PrxII andTNKS1.

In FIGS. 56 and 57. PrxII WT and G116V mutants showed the same levels ofexpression and peroxidase activity, however, as shown in FIG. 53, thePrxII G116V mutant did not block the inhibition of TNKS activity by H₂O₂but PrxII WT completely blocked the inhibition.

In addition, in Lanes 2 and 3 of FIG. 58, it was indicated that thePrx-SO_(2/3) blot completely peroxidated the endogenous 2-Cys Prx uponH₂O₂ treatment, whereas the exogenous PrxII with a C-terminal Myc tag(PrxII-Myc) was resistant to peroxidation.

In fact, as a result of the analysis of the activity of 2-Cys Prx exvivo using a recombinant enzyme, as shown in FIG. 58, the PrxII-Mycenzyme showed a strong peroxidation enzyme activity without any sign ofperoxidation, unlike the wild-type PrxII enzyme.

Since it is known that the C-terminal modification of the PrxII proteinconfers resistance to peroxidation, it predicted that the addition ofthe Myc tag to the C-terminus may result in a similar structural changein PrxII, which is converted to a peroxidation resistant form.

In addition, colony forming assays were performed to determine thebiological significance of the PrxII-TANKS interaction.

In FIG. 59, the colony formation of APC-mutant SW480 cells inhibited byPrxII deficiency was fully recovered by ectopic expression of PrxII WT,but the colony formation of the G116V mutant was not fully recovered.

From these results, it confirmed that the bound PrxII can preventoxidative inactivation of tankyrase by removing H₂O₂ from tankyrase,which is important for the growth of APC-mutant CRC cells.

<Example 14> Analysis of PrxII Expression in Human CRC Tissues

As shown in FIG. 60, as a result of the gene expression analysis of theCancer Genome Atlas (TCGA) database1, it confirmed that PrxII expressionwas significantly higher than normal colorectal tissue in tumorspecimens of colorectal adenocarcinoma patients and that the expressionof PrxII (i.e., the closest homologous protein) showed no suchdifference. In FIG. 61, an increased PrxII expression was observed inall tumor steps.

As shown in FIG. 62, the immunohistochemistry performed using CRC tissuearray showed PrxII levels approximately 2 times higher in CRC tissuescompared to normal tissues. From these results, it confirmed thatspecific PrxII induction may be a prerequisite for CRC expansion.

<Example 15> Regulation of PrII Activity by Compound 6 (Conoidin A)

Based on this assay, a cell-permeable compound of Compound 6 calledConoidin A was tested to evaluate the possibility of treating human CRCby inhibiting PrxII.

In the in vitro Prx assay of FIG. 63, it was found that Compound 6(Conoidin A) inhibited human PrxII activity by about 85% (214.9±24.3nmol min⁻¹ for the control, 38.8±17.1 nmol min⁻¹ for the group treatedwith Conoidin A), and inhibited PrxI activity by about 50%.

In comparison, the initial rate of PrxI activity was not affected byCompound 6 (Conoidin A) (263.7±45.4 nmol min⁻¹ for the control, 241.7±21nmol min⁻¹ for the group treated with Conoidin A).

<Example 16> Colony Forming Assay by Treatment with Compound 6 (ConoidinA)

The colony forming assay in FIG. 64 showed that Compound 6 (Conoidin A)treatment sufficiently inhibited the proliferation of HT29 and SW480cells but not RKO cells. This results suggest that the therapeuticpotential of PrxII inhibition to selectively target human APC-mutant CRCcells.

When Compound 6 (Conoidin A) was intraperitoneally injected into micewith a tumor xenograft derived from HT29, Compound 6 (Conoidin A)treatment significantly delayed tumor growth compared to the control, asshown in FIGS. 65 and 66.

From these results, it was indicated that PrxII may be a new targettreatment for human CRC, and a compound that inhibits PrxII, such asCompound 6 (Conoidin A), can be a new therapeutic drug for human CRC, asshown in the schematic diagram of FIG. 67.

Hereinafter, the synthesis method of Compound-1 to Compound-5 will bedescribed in Synthesis Examples. Compound-6 is Conoidin A and is widelysold by Candia Thamtech Company Limited: Shanghai YuLue Chemical Co.,Ltd., etc.

<Synthetic Example 1> Synthesis of2,3-bis(bromomethyl)-6-methoxyquinoxaline 1,4-dioxide (Compound-1)

Step 1

4-Methoxy-2-nitroaniline (1.01 g, 5.89 mmol) was added to a 20%KOH/ethanol solution (35 mL) prepared in advance and stirred. About 30mL of 12% NaOCl solution was added thereto in an ice bath and warmed toroom temperature and the solution was stirred until the startingmaterial disappeared.

The solid generated was filtered and washed with cold ethanol. Thethus-obtained yellow solid was subjected to a recrystallization processusing a solution (water:ethanol=1:3) (0.65 g, 66%).

¹H-NMR (400 MHz, CDC₃) δ 7.47-6.40 (m, 3H), and 3.90 (s, 3H).

Step 2

6-Methoxybenzo[c][1,2,5]oxadiazole 1-oxide (1.00 g, 6.02 mmol) wasstirred with trimethylamine (18.06 mmol). Pyrrolidine (0.78 g, 10.83mmol) and methylethylketone (0.65 g, 9.03 mmol) were added dropwise inan ice bath.

The termination of reaction was confirmed about one hour afterincreasing the temperature to room temperature (TLC analysis conditions,n-hexane:EtOAc=2:1). The reaction solution was filtered and then washedwith cold ethanol to obtain a brown solid (1.06 g, 80%). In this step,the reaction was proceeded to the next step without furtherpurification.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.36 (d, J=9.6 Hz, 1H), 7.78 (d, J=2.0 Hz,1H), 7.49 (dd, J=9.0 Hz, 3.0 Hz, 1H) 3.97 (s, 3H), 2.59 (s, 3H), and2.56 (s, 3H).

Step 3

6-Methoxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.54 mmol) wasdissolved in 1,4-dioxide, and bromine (12.71 mmol) was added dropwise tothe mixture.

The reaction solution was heated up to 90° C. and reacted for about twohours until the spot of the starting material disappeared. Inparticular, the reaction progress was determined using TLC analysis(n-hexane:EtOAc=1:2).

After completion of the reaction, an aqueous NaHCO₃ solution was addedthereto and the mixture was extracted with ethyl acetate. Then, theresulting organic layer was washed with brine, dried over MgSO₄,filtered, and concentrated to obtain a product in a mixed state. Thisproduct was columned using MPLC (n-hexane:EtOAc=3:1) to obtainCompound-1 (1.15 g, 65%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.39 (d, J=9.6 Hz, 1H), 7.81 (d, J=2.4 Hz),7.59 (dd, J=7.4 Hz, 2.2 Hz), 5.06 (s, 4H), and 4.01 (s, 3H); ¹³C-NMR(100 MHz, DMSO-d₆) δ 162.9, 124.4, 122.2, 99.5, 57.0, 23.5, and 23.4;HRMS (ESI): m/z 377.9038 [M+H]⁺ (calcd for C₁₁H₁₀Br₂N₂O₃ ⁺ 378.9170)

<Synthetic Example 2> Synthesis of2,3-bis(bromomethyl)-6-ethoxyquinoxaline 14-dioxide) (Compound-2)

Step 1

A subdivision of potassium carbonate (6.73 g, 48.66 mmol) was addedwhile stirring 4-amino-3-nitrophenol (5.00 g, 32.44 mmol) andbromoethane (5.30 g, 48.66 mmol) in DMF solvent.

Then, the reaction vessel was heated to 90° C. and the reaction wasperformed for about three hours and observed until the starting materialdisappeared (TLC analysis conditions, n-hexane:EtOAc=3:1).

After completion of the reaction, the reaction solution was cooled toroom temperature and added to ice water. The solid generated wasfiltered to obtain a red solid in a mixed state. The resultant waspurified through a column (n-hexane:EtOAc=10:1) and obtained4-ethoxy-2-nitroaniline (4.25 g, 72%).

¹H-NMR (400 MHz, CDCl₃) δ 7.55 (bs, 1H), 7.07 (d, J=8 Hz, 1H), 6.77 (d,J=8 Hz, 1H), 5.89 (bs, 2H), 4.01 (m, 2H), and 1.41 (t, J=6.0 Hz, 3H).

Step 2

4-Ethoxy-2-nitroaniline (2.00 g, 10.98 mmol) was added to a 20%KOH/ethanol solution (60 mL) prepared in advance and stirred.

About 50 mL of a 12% NaOCl solution was added dropwise thereto in an icebath, and the mixture was warmed to room temperature and stirred untilthe starting material disappeared. The solid generated was filtered andwashed with cold ethanol. The thus-obtained yellow solid was subjectedto a recrystallization process using a solution (water:ethanol=1:3solution) (1.85 g, 94%).

¹H-NMR (400 MHz, CDCl₃) δ 7.49-6.34 (m, 3H), 4.08 (m, 2H), and 1.48 (t,J=8.0 Hz, 3H)

Step 3

6-Ethoxybenzo[c][1,2,5]oxadiazole 1-oxide) (1.50 g, 8.33 mmol) wasstirred with trimethylamine (2.52 g, 24.98 mmol).

Pyrrolidine (1.08 g, 14.99 mmol) and methylethylketone (0.90 g, 12.49mmol) were added dropwise in an ice bath.

The reaction was terminated about one hour after the reactiontemperature was raised to room temperature (TLC analysis conditions,n-hexane:EtOAc=2:1). The reaction solution was filtered and washed withcold ethanol to obtain a brown solid (1.11 g, 57%). In this step, thereaction was proceeded to the next step without further purification.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.51 (d, J=9.6 Hz, 1H), 7.90 (d, J=2.4 Hz,1H), 7.38 (dd, J=9.4, 2.6 Hz, 1H), 4.24 (m, 2H), 2.74 (s, 3H), 2.71 (s,3H), and 1.51 (t, J=6.8 Hz, 3H).

Step 4

6-Ethoxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.27 mmol) wasdissolved in 1,4-dioxide, and bromine (12.71 mmol) was added dropwise tothe mixture.

The reaction solution was heated up to 90° C. and reacted for about twohours until the spot of the starting material disappeared. Inparticular, the reaction progress was determined using TLC analysis(n-hexane:EtOAc=1:2). After completion of the reaction, an aqueousNaHCO₃ solution was added thereto and the mixture was extracted withethyl acetate.

Then, the resulting organic layer was washed with brine, dried overMgSO₄, filtered, and concentrated to obtain a product in a mixed state.This product was columned using MPLC (n-hexane:EtOAc=3:1) to obtainCompound-2 (0.98 g, 61%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.52 (d, J=9.2 Hz, 1H), 7.90 (d, J=2.4 Hz,1H), 7.42 (dd, J=9.6, 2.8 Hz), 4.90 (s, 4H), 4.24 (m, 2H), and 1.52 (t,J=7.2 Hz, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ 163.2, 122.5, 122.1, 101.2,58.0, 23.9 and 13.9; HRMS (ESI): m/z 391.9194 [M+H]⁺ (calcd forC₁₂H₁₂Br₂N₂O₃ ⁺ 392.9155)

<Synthetic Example 3> Synthesis of2,3-bis(bromomethyl)-6-isopropoxyquinoxaline 1,4-dioxide (Compound-3)

Step 1

A subdivision of potassium carbonate (6.73 g, 48.66 mmol) was addedwhile stirring 4-amino-3-nitrophenol (5.00 g, 32.44 mmol) and2-bromopropane (5.98 g, 48.66 mmol) in DMF solvent.

Then, the reaction vessel was heated to 90° C. and the reaction wasperformed for about five hours and observed until the starting materialdisappeared (TLC analysis conditions, n-hexane:EtOAc=3:1).

After completion of the reaction, the reaction solution was cooled toroom temperature and added to ice water. Then, an extraction wasperformed with diethyl ether until the spot in the aqueous layerdisappeared. The resulting organic layer was washed with brine, driedover MgSO₄, and concentrated to obtain a red solid.

This was purified through a column (n-hexane:EtOAc=10:1) and obtained4-isopropoxy-2-nitroaniline (5.90 g, 93%).

¹H-NMR (400 MHz, CDCl₃) δ 7.51 (bs, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.75(d, J=8.0 Hz, 1H), 5.88 (bs, 2H), 4.46 (m, 1H), and 1.33 (d, J=6.0 Hz,6H).

Step 2

4-Isopropoxy-2-nitroaniline (2.00 g, 10.19 mmol) was added to a 20%KOH/ethanol solution (60 mL) prepared in advance and stirred.

About 50 mL of a 12% NaOCl solution was added dropwise thereto in an icebath, and the mixture was warmed to room temperature and stirred untilthe starting material disappeared. The solid generated was filtered andwashed with cold ethanol. The thus-obtained yellow solid was subjectedto a recrystallization process using a solution (water:ethanol=1:3)(1.93 g, 97%).

¹H-NMR (400 MHz, CDCl₃) δ 7.48-6.34 (m, 3H), 4.60 (m, 1H), and 1.41 (d,J=6.0 Hz, 6H).

Step 3

6-Isopropoxybenzo[c][1,2,5]oxadiazole 1-oxide) (1.50 g, 7.72 mmol) wasstirred with trimethylamine (2.34 g, 23.17 mmol).

Pyrrolidine (1.00 g, 13.90 mmol) and methylethylketone (0.84 g, 11.59mmol) were added dropwise in an ice bath. The reaction was terminatedabout one hour after the temperature was increased to room temperature(TLC analysis conditions, n-hexane:EtOAc=2:1). The reaction solution wasfiltered and washed with cold ethanol to obtain a brown solid (1.49 g,78%). In this step, the reaction was proceeded to the next step withoutfurther purification.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.50 (d, J=9.6 Hz, 1H), 7.91 (d, J=2.4 Hz,1H), 7.34 (dd, J=9.4 Hz, 2.6 Hz, 1H) 4.81 (s, 1H), 2.73 (s, 3H), 2.71(s, 3H), and 1.44 (d, J=6.0 Hz, 3H).

Step 4

6-Isoproxy-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.03 mmol) wasdissolved in 1,4-dioxide, and bromine (12.71 mmol) was added dropwise tothe mixture.

The reaction solution was heated up to 90° C. and reacted for about twohours until the spot of the starting material disappeared. Inparticular, the reaction progress was determined by TLC analysis(n-hexane:EtOAc=1:2).

After completion of the reaction, an aqueous NaHCO₃ solution was addedthereto and the resultant was extracted with ethyl acetate. Theresulting organic layer was washed with brine, dried over MgSO₄,filtered, and concentrated to obtain a product in a mixed state. Thisproduct was columned using MPLC (n-hexane:EtOAc=3:1) to obtainCompound-3 (0.69 g, 42%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.52 (d, J=9.2 Hz, 1H), 7.90 (d, J=2.4 Hz,1H), 7.42 (dd, J=9.6, 2.8 Hz), 4.90 (s, 4H), 4.24 (m, 2H), and 1.52 (t,J=7.2 Hz, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ 162.9, 122.70, 105.7, 73.0,23.9 and 21.8; HRMS (ESI): m/z 405.9351 [M+H]⁺ (calcd for C₁₃H₁₄Br₂N₂O₃⁺ 406.9458)

<Synthetic Example 4> Synthesis of6-(allyloxy)-2,3-bis(bromomethyl)quinoxaline 1,4-dioxide) (Compound-4)

Step 1

A subdivision of potassium carbonate (6.73 g, 48.66 mmol) was addedwhile stirring 4-amino-3-nitrophenol (5.00 g, 32.44 mmol) and allylbromide (5.89 g, 48.66 mmol) in DMF solvent.

Then, the reaction vessel was heated to 90° C. and the reaction wasperformed and observed for about three hours until the starting materialdisappeared (TLC analysis conditions, n-hexane:EtOAc=3:1).

After completion of the reaction, the reaction solution was cooled toroom temperature and added to ice water. Then, an extraction wasperformed with diethyl ether until the spot in the aqueous layerdisappeared. The resulting organic layer was washed with brine, driedover MgSO₄, and concentrated to obtain a red solid.

This was purified through a column (n-hexane:EtOAc=10:1) and obtained4-(allyloxy)-2-nitroaniline (4.44 g, 71%).

¹H-NMR (400 MHz, CDCl₃) δ 7.52 (bs, 1H), 7.00 (d, J=7.6 Hz, 1H), 6.73(d, J=8.0 Hz, 1H), 6.06 (m, 1H), 5.60 (bs, 2H), 5.47 (d, J=16.0 Hz, 1H),5.34 (d, J=10.4 Hz, 1H), and 4.70 (d, J=5.2 Hz, 2H).

Step 2

4-Allyloxy-2-nitroaniline (2.00 g, 10.30 mmol) was added to a 20%KOH/ethanol solution (60 mL) prepared in advance and stirred.

About 50 mL of a 12% NaOCl solution was added dropwise thereto in an icebath, and the mixture was warmed to room temperature and stirred untilthe starting material disappeared. The solid generated was filtered andwashed with cold ethanol. The thus-obtained yellow solid was subjectedto a recrystallization process using a solution (water:ethanol=1:3solution (1.96 g, 99%).

¹H-NMR (400 MHz, CDCl3) δ 7.76-6.69 (m, 3H), 6.06 (m, 1H).

Step 3:

6-(Allyloxy)benzo[c][1,2,5]oxadiazole 1-oxide (1.50 g, 7.81 mmol) wasstirred with trimethylamine (2.36 g, 23.42 mmol).

Pyrrolidine (1.01 g, 14.05 mmol) and methylethylketone (0.84 g, 11.71mmol) were added dropwise thereto. The termination of reaction wasconfirmed about one hour after increasing the temperature to roomtemperature (TLC analysis conditions, n-hexane:EtOAc=2:1). The reactionsolution was filtered and then washed with cold ethanol to obtain abrown solid (0.96 g, 50%). In this step, the reaction was proceeded tothe next step without further purification.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.38 (d, J=9.6 Hz, 1H), 7.82 (d, J=2.4 Hz,1H), 7.52 (dd, J=9.4, 2.4 Hz, 1H), 6.10 (m, 1H), 5.57 (dd, J=17.2, 1.6Hz, 1H), 5.34 (dd, J=10.6, 1.6 Hz, 1H), 2.59 (s, 3H), 2.57 (s, 3H).

Step 4:

6-(Allyloxy)-2,3-dimethylquinoxaline 1,4-dioxide (1.00 g, 4.06 mmol) wasdissolved in 1,4-dioxide, and bromine (12.71 mmol) was added dropwise tothe mixture.

The reaction solution was heated up to 90° C. and reacted for about twohours until the spot of the starting material disappeared. Inparticular, the reaction progress was determined using TLC analysis(n-hexane:EtOAc=1:2). After completion of the reaction, an aqueousNaHCO₃ solution was added thereto and the resultant was extracted withethyl acetate. Then, the resulting organic layer was washed with brine,dried over MgSO₄, filtered, and concentrated to obtain a product in amixed state.

This product was columned using MPLC (n-hexane:EtOAc=3:1) to obtainCompound-4 (0.72 g, 44%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.25 (d, J=9.6 Hz, 1H), 7.90 (m, 2H), 5.87(m, 1H) 5.23-5.17 (m, 2H), 4.92 (s, 4H), and 4.61 (d, J=4.8 Hz, 2H);¹³C-NMR (100 MHz, DMSO-d₆) δ 167.0, 134.5, 122.3, 122.1, 117.5, 105.0,70.1, and 23.9 HRMS (ESI): m/z 403.9194 [M+H]⁺ (calcd for C₁₃H₁₂Br₂N₂O₃⁺ 404.9279)

<Synthetic Example 5> Synthesis of2,3-bis(bromomethyl)-6-(prop-2-ynyloxy)quinoxaline 1,4-dioxide(Compound-5)

Step 1:

A subdivision of potassium carbonate (6.73 g, 48.66 mmol) was addedwhile stirring 4-amino-3-nitrophenol (5.00 g, 32.44 mmol) and propargylbromide (5.79 g, 48.66 mmol) in DMF solvent. Then, the reaction vesselwas heated to 90° C. and the reaction was performed and observed forabout three hours until the starting material disappeared (TLC analysisconditions, n-hexane:EtOAc=3:1).

After completion of the reaction, the reaction solution was cooled toroom temperature and added to ice water. Then, an extraction wasperformed diethyl ether until the spot in the aqueous layer disappeared.The resulting organic layer was washed with brine, dried over MgSO₄, andconcentrated to obtain a red solid.

This finally confirmed 2-nitro-4-(prop-2-yn-1-yloxy)aniline (3.95 g,63%) through a column (n-hexane:EtOAc=10:1).

¹H-NMR (400 MHz, CDCl₃) δ 7.69 (bs, 1H), 7.13 (d, J=8.0 Hz, 1H), 6.78(d, J=8.0 Hz, 1H), 5.91 (bs, 2H), 4.67 (d, J=4.0 Hz, 2H), and 2.55 (t,J=2.0 Hz, 1H).

Step 2:

2-Nitro-4-(prop-2-yn-1-yloxy) aniline (2.00 g, 10.41 mmol) was added toa 20% KOH/ethanol solution (60 mL) prepared in advance and stirred.About 50 mL of 12% NaOCl solution was added thereto in an ice bath andthe mixture was warmed to room temperature and stirred until thestarting material disappeared.

The solid generated was filtered and washed with cold ethanol. Thethus-obtained yellow solid was subjected to a recrystallization processusing a solution (water:ethanol=1:3) (1.96 g, 99%).

¹H-NMR (400 MHz, CDCl₃) δ 7.81-6.81 (m, 3H), 4.98 (d, J=2.0 Hz, 2H), and3.34 (s, 1H).

Step 3:

6-(Prop-2-yn-1-yloxy)benzo[c][1,2,5]oxadiazole 1-oxide) (1.50 g, 7.89mmol) was stirred with trimethylamine (2.39 g, 23.66 mmol).

Pyrrolidine (1.02 g, 14.20 mmol) and methylethylketone (0.85 g, 11.83mmol) were added dropwise in an ice bath. The termination of reactionwas confirmed about one hour after increasing the temperature to roomtemperature (TLC analysis conditions, n-hexane:EtOAc=2:1). The reactionsolution was filtered and then washed with cold ethanol to obtain abrown solid (0.96 g, 51%). In this step, the reaction was proceeded tothe next step without further purification.

¹H-NMR (400 MHz, DMSO-d₆) δ 8.38 (d, J=9.6 Hz, 1H), 7.92 (d, J=2.4, 1H),7.51 (dd, J=9.4, 2.6 Hz, 1H), 5.06 (d, J=2.4 Hz, 2H), 3.71 (t, J=2.4 Hz,1H), 2.59 (s, 1H), 2.57 (s, 1H).

Step 4:

A 2,3-dimethyl-6-(prop-2-ynyloxy)quinoxaline 1,4-dioxide (1.00 g, 4.09mmol) was dissolved in 1,4-dioxide, and bromine (12.71 mmol) was addedthereto.

The reaction solution was heated up to 90° C. and reacted for about twohours until the spot of the starting material disappeared. Inparticular, the reaction progress was determined using TLC analysis(n-hexane:EtOAc=1:2). After completion of the reaction, an aqueousNaHCO₃ solution was added thereto and the resultant was extracted withethyl acetate.

Then, the resulting organic layer was washed with brine, dried overMgSO₄, filtered, and concentrated to obtain a product in a mixed state.This product was columned using MPLC (n-hexane:EtOAc=3:1) to obtainCompound-5 (0.68 g, 41%).

¹H-NMR (400 MHz, DMSO-d₆) δ 8.34 (d, J=9.6 Hz, 1H), 8.06 (m, 2H); 5.00(s, 4H), 4.86 (d, J=2.4 Hz, 2H) and 3.51 (t, J=2.4 Hz, 1H); ¹³C-NMR (100MHz, DMSO-d₆) δ 164.8, 122.2, 120.6, 106.3, 80.0, 78.8, 58.8 and 23.1;HRMS (ESI): m/z 401.9038 [M+H]⁺ (calcd for C₁₃H₁₀Br₂N₂O₃ ⁺ 402.9465)

<Example 17> Analysis of Regulation of PrxII Activity by Compound-1 toCompound-5

To evaluate the possibility of treating human CRC by inhibiting PrxII,cell-permeability of Compound-1 to Compound-5 were tested.

In the graphs of FIGS. 72 and 73, it confirmed that Compound-1 toCompound-5 synthesized according to an embodiment of the presentinvention significantly inhibited PrxII activity.

<Example 18> Colony Forming Assay by Compound-1 to Compound-5

FIG. 74 shows a graph illustrating the results of the number of coloniesafter treatment of Compound-1 to Compound-5 in RKO cells. FIG. 75 showsthe results of colony forming assay obtained by culturing RKO cellsafter treating with Compound-1 to Compound-5 and Conoidin A(Compound-6).

FIG. 76 shows a graph illustrating the results of the number of coloniesafter treatment of Compound-1 to Compound-5 in HT29 cells. FIG. 77 showsthe results of colony forming assay obtained by culturing HT29 cellsafter treating with Compound-1 to Compound-5 and Conoidin A(Compound-6).

These results showed that the treatment with Compound-1 to Compound-5sufficiently inhibits the proliferation of HT29 cells but not RKO cells.From these results it predicted that PrxII can be a new targetedtreatment for human CRC, and compounds that inhibit PrxII, such asConoidin A (Compound-6) and Compound-1 to Compound-5, can be newtherapeutics for human CRC.

<Reference Example 1> Analysis of The Cancer Genome Atlas (TCGA)

The expression of PrxI and PrxII in CRC and normal colorectal tissuesamples was measured using microarray data from The Cancer Genome Atlas(TCGA) project (https://tcga-data.nci.nih.gov).

To illustrate, mRNA expression data were generated using the AgilentG4502A microarray platform and then processed and normalized asdescribed previously. For the analysis of gene expression data, 155 CRCtissue samples and 26 normal colorectal tissue samples were included.

<Reference Example 2> Immunoblotting and Immunoprecipitation

The intestinal lumen was washed with ice-filled phosphate bufferedsaline (PBS) using a syringe with a flat needle and cut longitudinally.Intestinal sections without polyps were excised along with polyps forimmunoblotting analysis.

Tissues were homogenized in HEPES-buffered saline containing 10%glycerol, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 5 mM Na₃VO₄, 5 mM NaF, 1 mMAEBSF, aprotinin (5 μg/mL), and leupetin (5 μg/mL) using a Douncehomogenizer.

The cultured cells were rinsed once with ice-cold PBS and dissolved inlysis buffer containing 20 mM HEPES (pH 7.0), 1% Triton X-100, 150 mMNaCl, 10% glycerol, 1 mM EDTA, 2 mM EGTA, 1 mM DTT, 5 mM Na₃VO₄, 5 mMNaF, 1 mM AEBSF, aprotinin (5 μg/mL), and leupetin (5 μg/mL).

Homogenates and cell lysates were centrifuged at 15,000×g for 15 minutesand protein concentrations were examined by Bradford assay (Pierce).Protein samples were mixed with an SDS sample buffer and boiled for 5minutes.

The proteins were isolated by SDS-PAGE and transferred to nitrocellulosemembranes by electroblotting for one hour. The membranes were blockedwith 5% bovine serum albumin (BSA) or 5% dried skim milk for two hoursin Tris buffered saline containing 0.05% (v/v) Tween-20 (TBST) and thenincubated at a constant temperature in blocking buffer along with anappropriate primary antibody at room temperature for two hours.

Then, the membranes, after washing three times with TBST, were incubatedwith a horseradish peroxidase-conjugated secondary antibody (AmershamBiosciences) in blocking buffer. The immune-reactive bands were detectedby a chemiluminescence kit (AbFrontier. Korea) and quantified with aLAS-3000 imaging system (Fuji Film, Japan).

If necessary, the membranes were removed by shaking at 37° C. for 60minutes in 67 mM Tris (pH 6.7), 2% SDS, and 100 mM β-mercaptoethanol,and re-examined with an appropriate general antibody (pan-antibody).

For immunoprecipitation, purified cell lysates (0.5-1 mg of proteins)were removed in advance with 30 μL of protein-A/G Sepharose 4 Fast Flowbeads (Amersham Biosciences) for one hour.

Supernatants (cultures floating on the surface) were incubated with 3 μgof an appropriate antibody overnight, and then, precipitated by mixingwith 30 μL of protein-A/G beads at 4° C. for three hours.

Then, the beads were washed three times with 1 mL of lysis buffer,followed by in vitro PARP analysis or immunoblotting.

<Reference Example 3>β-Catenin/TCF Transcription Reporter Assay

SW480 cells were seeded in a 12-well plate and transfected withpTOPflash or pFOPflash plasmid. To normalize transfection efficiency,cells were transfected with pRL-TK renilla Luciferase control plasmid.

After the transfection, cells were incubated for 24 hours in a completemedium and then lysed with reporter lysis buffer. Luciferase activitywas measured three times by Dual-Luciferase reporter assay (Promega).

Data were reported as fold induction compared to the control siRNA afternormalization of transfection efficiency. As used herein, “foldinduction” refers to a ratio of experimental activity to controlactivity.

<Reference Example 4> RNA Sequence Analysis

Four samples of siRNA-transfected HT29 cells were prepared forhigh-throughput mRNA sequencing. 1 μg of RNA was extracted from eachsample and an mRNA library (an insert size of about 300 bp) wasconstructed using TruSeq RNA Library Preparation kit v2 (Illumina).

Paired-end transcriptome sequencing (101 bp read length) was performedusing Illumina HiSeq 2500. The number of reads for each sample rangedfrom 69.4 million to 74.8 million. The sequencing data were deposited inthe GEO database (Accession Number GSE81429).

After standard quality testing and trimming with FastQC andFastx-toolkit, RNA sequence data was aligned to the human genome (hg19of UCSC) using MapSplice v2.1.7. The mapping rate of reads was between96.5% and 96.8%, and RSG v1.2.12 was used to estimate the amount oftranscriptome of refGene mRNA.

Differentially expressed genes (DEG) were identified using DESeq2 withan FDR cutoff of 0.05.

<Reference Example 5> In Vitro PARP Assay

Immunocomplex-bound beads were incubated at 25° C. for 30 minutes in 40μL of assay buffer (50 mM Tris-HCl, pH 8.0, 4 mM MgCl₂) containing 4 μCiof γ-³²[P]-NAD⁺. The reaction was stopped by adding 2×SDS sample buffer.The sample was boiled and separated using an SDS denaturing gel.

The gel was dried under vacuum and radiographed on an imaging plate.Radioactivity recorded on the plate was read and quantified by FujifilmBio-imaging Analyzer System (BAS)-3000.

<Reference Example 6> Plasmid Construction and Site-Directed Mutagenesis

A plasmid including the full-length complementary DNA of human tankyrase1 (TNKS1) was purchased from Open Biosystems (mRNA accession number,BC098394). The entire sequence of tankyrase-1 was PCR-amplified usingthe forward and reverse primers:

(SEQ ID NO: 11) 5′-ATAAGAATGCGGCCGCGGCGGCGTCGCGTCGCTC-3′ and(SEQ ID NO: 12) 5′-GAAGATCTCTAGGTCTTCTGCTCTG-3′

Then, the amplified PCR products was inserted into p3×FLAG CMV9 vectorto generate FLAG-tagged tankyrase 1 (TNKS1). For domain mappingexperiments, various tankyrase 1 fragments were PCR-amplified andsubcloned into the p3×FLAG CMV9 vector using the following forward andreverse primers:

amino acid residue 1-158, (SEQ ID NO: 13)5′-ATAAGAATGCGGCCGCGGCGGCGTCGCGTCGCTC-3′ and (SEQ ID NO: 14)5′-GAAGATCTCTAGGCCGCCTCGGGGCTCTC-3′; amino acid residue 158-595,(SEQ ID NO: 15) 5′-ATAAGAATGCGGCCGCCGGAGTTAGCAGCACAGCAC-3′ and(SEQ ID NO: 16) 5′-GAAGATCTCTACAAAGCAGTCTGACCAAGGG-3′;amino acid residue 596-1022, (SEQ ID NO: 17)5′-ATAAGAATGCGCCGCGCATAGAGCCGCCCTAGCAGG-3′ and (SEQ ID NO: 18)5′-GAAGATCTCTATCCTTCCTTCCTTTCTGTTCC-3′; amino acid residue 1023-1327,(SEQ ID NO: 19) 5′-ATAAGAATGCGGCCGCAGAAGTTGCTGGTCTTGAC-3′ and(SEQ ID NO: 20) 5′-GAAGATCTCTAGGTCTTCTGCTCTG-3′

The E. coli expressing plasmid for GST-TNKS1 (1023-1327) was kindlyprovided by Chang-Woo Lee (School of Medicine, Sungkyunkwan University).

Myc-tagged siRNA-resistant PrxII WT, and a retroviral vector (pQ-CXIX)expressing a C172S single mutant and a C51/172S double mutant wereprepared as described above. Site-directed mutagenesis for amino acidsubstitutions was performed using a QuikChange kit (Stratagene).

The double-stranded primers for Cys-Ser substitution in tankyrase 1 areas follows:

in the case of a C1163S mutant, (sense) (SEQ ID NO: 21)5′-GTTGAGGGAGCGGTTCTCCCACCGACAGAAGGAAG-3′;in the case of a C1234S mutant, (sense) (SEQ ID NO: 22)5′-GGAGGAGGAACAGGCTCCCCTACACACAAGGAC-3′; in the case of a C1242S mutant,(sense) (SEQ ID NO: 23) 5′-CACAAGGACAGGTCATCCTATATATGTCACAGAC-3′;in the case of a C1245S mutant, (sense) (SEQ ID NO: 24)5′-CAGGTCATGCTATATATCTCACAGACAAATGCTCTTC-3′;in the case of a C1252S mutant, (sense) (SEQ ID NO: 25)5′-GACAAATGCTCTTCTCTAGAGTGACCCTTGGG-3′

The double-stranded primers for Gly-Val substitution of human PrxII areas follows:

in the case of a G9V mutant, (sense) (SEQ ID NO: 26)5′-GCGCGCATCGTAAAGCCAGCCCCTG-3′; in the case of a G23V mutant, (sense)(SEQ ID NO: 27) 5′-GCGGTGGTTGATGTCGCCTTCAAAG-3′;in the case of a G116V mutant, (sense) (SEQ ID NO: 28)5′-CTGAGGATTACGTCGTGCTGAAAAC-3′.

A retroviral vector pQ vector expressing β-catenin S37A mutants wasprepared by PCR subcloning from the pBI-EGFP-β-catenin (S37A) structuredescribed previously. All of the structures and mutations were confirmedby nucleotide sequencing.

<Reference Example 7> Zinc Determination

Zinc ions were observed using 4-(2-pyridylazo) resorcinol (PAR) in anaqueous solution.

The glutathione S-transferase (GST)-fused TNKS1 PARP (1023-1327) proteinwas expressed in E. coli grown in LB medium supplemented with 100 μMZnCl₂, and purified by affinity chromatography using GlutathioneSepharose 4B Fast Flow beads according to the manufacturer's protocol(GE Healthcare Life Sciences).

The purity (>99.5%) of a GST-TNKS1 PARP protein was confirmed by theconcentration measurement method, and then, broadly dialyzed inChelex100 treated buffer containing 25 mM HEPES (pH 7.0) and 2 mM DTT toremove unbound zinc ions.

The GST-TANK1 PARP protein was incubated with 500 μM H₂O₂ for 30 minutesin 200 μL of 40 mM HEPES (pH 7.0) reaction buffer containing 0.1 mM PAR.The formation of a PAR2-Zn²⁺ complex was monitored at 500 nm with aUV/VIS spectrophotometer (Agilent).

The total zinc content of the purified GST-TNKS1 PARP protein used inthe assay was determined by adding 0.5 mM p-chloromercuribenzoic acid toa reaction mixture.

<Reference Example 8> Peroxiredoxin Assay

The peroxidase assay was performed in a reaction mixture (200 μL)containing 250 μM NADPH, 1.5 μM yeast TR, 3 μM yeast Trx, a recombinanthuman Prx (PrxI (4.6 μM) and PrxII (16.4 μM)), and 50 μM HEPES (pH 7.0)containing 1 mM EDTA, and 200 M H₂O₂.

The mixture (minus H₂O₂) was preincubated for 5 minutes in the presenceor absence of Compounds 1 to 6 (100 μM), and then, the reaction wasinitiated by adding H₂O₂. NADPH oxidation was monitored at 30° C. for 5minutes as the absorbance decreased near 340 nm using an Agilent UV8453spectrophotometer (Hewlett Packard. USA). The initial reaction rate wascalculated using the linear portion of the curve and indicated as theamount of oxidized NADPH per minute.

<Reference Example 9> Histology, Immunohistochemistry, andImmunofluorescence Staining

12-Week-old male mice were anesthetized by inhalation of isoflurane gas(N₂O:O₂/70%: 30%) and subjected to transcardiac perfusion-fixation withheparinized saline containing 3.7% formaldehyde.

Then, the intestine was excised and cut into two parts, the smallintestine and the colon, both of which were opened vertically and foldedoutward. The folded intestine was paraffin inserted and sectioned by arotary microtome (Leica RM2255).

Three serial tissue sections with a thickness of 10 μm were stained withhematoxylin and eosin (HE). The folded intestine was immediately buriedin OCT medium and frozen in dry ice. A cryostatt microtome, cryotome,was used to cut the tissue into 10 μm cross sections.

Cryotome is a microtome that handles frozen tissue. A microtome is amachine that cuts samples into pieces of a certain thickness to preparespecimens for microscopic observation. The samples were placed onSuperfrost Plus slides (Surgipath Medical Inc. UK) and dried at roomtemperature and maintained at −80° C. until thawed for immunostaining.

Paraffin sections were dewaxed with xylene and rehydrated in ethanol forimmunohistochemistry. Then, antigen retrieval was performed by boilingthe sections in sodium citrate buffer (pH 6.0).

Tissue sections were incubated at 4° C. with an anti-Ki-67 antibody (a1:200 dilution) and an affinity-purified anti-PrxII antibody (a 1:500dilution) for 48 hours. After washing three times with PBS, the sectionswere incubated with a peroxidase-conjugated secondary antibody andstained with a 3,3′-diaminobenzidine (DAB) substrate solution.

Nuclei were further stained with hematoxylin. DAB staining images wereobtained and quantified using HistoFAXS Tissue Analysis System(TissueGnostics, USA). For immunofluorescence staining, paraffin orfrozen sections were blocked at room temperature for one hour with 5%normal rabbit serum (Vector Laboratories) in PBST (0.3% Triton X-100 inPBS).

Sections were incubated at 4° C. overnight with a primary antibody (a1:500 dilution for the anti-PrxII antibody and a 1:100 dilution for theanti-BrdU antibody). After washing several times with PBST, the sampleswere incubated at room temperature with the Alexa Fluor 568-conjugateddonkey anti-rabbit IgG antibody for 2 hours.

Sections were counterstained with 4′,6-diamidino-2 phenylindole (DAPI,Sigma-Aldrich) for 30 minutes, and mounted using Vectashield mountingmedium.

Fluorescence images were obtained at three random fields per tissuecross-section at 100× magnification using an LSM 51 Meta confocalmicroscope equipped with an argon and helium-neon laser (Carl Zeiss.Germany).

<Reference Example 10> Statistical Analysis

Unless otherwise specified, to determine statistical significance (Pvalue), the data was analyzed by Analysis of Variance (ANOVA) using theStudent's t-test for comparison between two groups, or using theTukey-HSD or Tukey test for comparison of multiple groups (SPSS 12.0K incase of Windows, SPSS, Ill., USA). P<0.05 was considered statisticallysignificant.

<Reference Example 11> Data Availability

The RNA sequencing data that supports the findings is deposited in theGene Expression Omnibus database (GEO DB) (Accession Number GSE81429).

<Reagent>

The anti-tubulin antibody (mouse monoclonal, B-5-1-2, 1:8,000, T5168)and the anti-FLAG antibody (mouse monoclonal, M2, 1:1,000, F3165) werepurchased from Sigma-Aldrich.

The antibodies for β-catenin (rabbit monoclonal, 6B3, 1:1,000, 9582),Axin1 (rabbit monoclonal, C76H11, 1:1,000, 2087), pS33/37pT41-β-catenin(rabbit polyclonal, 1:1,000, 9561), Axin2 (rabbit monoclonal, 76G6,1:1,000, 2151), GSK3β(rabbit monoclonal, 27C10, 1:1,000, 9315), β-actin(rabbit monoclonal, 13E5, 1:1,000, 4970), and cyclin D1 (rabbitpolyclonal, 1:1,000, 2922) were purchased from Cell SignalingTechnology.

The antibodies for c-Myc (rabbit polyclonal, 1:1,000, sc-788), Ubiquitin(mouse monoclonal, P4D1, 1:1,000, sc-8017), pY279/216-GSK3β (rabbitpolyclonal, 1:1,000, sc-135653), and tankyrase-½ (rabbit polyclonal,1:1,000, H-350) were purchased from Santa Cruz Biotechnology.

The antibodies for anti-active β-catenin (mouse monoclonal, 8E7,1:1,000, 05-665), anti-Myc (mouse monoclonal, 9E10, 1:1,000, 05-419),and anti-APC (mouse monoclonal, FE9, 1:1,000, ABC202) were purchasedfrom Millipore.

The antibodies for Alexa Fluor 568-conjugated donkey, anti-rabbit IgG(1:200, A-21206), and anti-β-TrCP (mouse monoclonal, 1B1D2, 1:1,000,37-3400) were purchased from Invitrogen.

The anti-PAR antibody that detects poly(ADP-ribose) chains (rabbitpolyclonal, 1:2,000, 551813) was purchased from BD Bioscience.

The anti-Ki-67 antibody (rabbit monoclonal, SP6, 1:200, MA5-14520) waspurchased from Thermo Fisher Scientific.

As described previously, rabbit polyclonal antibodies for PrxI(1:3,000), PrxII (1:3,000), and Prx-SO ⅔ (1:1,000) were produced.

Rabbit anti-PrxII antiserum was affinity-purified with a recombinantPrxII protein and agarose gel beads and used for immunofluorescence andproximity ligation assays (PLA).

Wnt3a was purchased from R & D Biosystems.

DuoLink in situ fluorescence reagent was purchased from Sigma-Aldrich.

TissueFocus Colorectal Tissue Microarray was purchased from OriGeneTechnologies (Rockville, USA).

Examples of the present invention described above have been describedwith reference to the embodiments shown in the drawings for ease ofunderstanding, however, these are merely exemplary, and it will beunderstood by those skilled in the art that various modifications andequivalent other examples are possible therefrom. Therefore, the truetechnical protection scope of the present invention should be defined bythe appended claims.

INDUSTRIAL APPLICABILITY

According to an embodiment of the invention, a composition, which cantreat or prevent colorectal cancer (CRC) and reduce colorectal polyps byinhibiting the enzyme activity of peroxiredoxin 2 (PrxII) via regulationof the redox system of colorectal cancer (CRC) cells, can be provided.

According to another embodiment of the invention, a composition, whichcan treat or prevent colorectal cancer (CRC) and reduce colorectalpolyps by reducing the interaction between peroxiredoxin 2 (PrxII) andtankyrase (TNKS) in the cytoplasm of APC-mutant cells, can be provided.

The invention claimed is:
 1. A method for improving or treatingcolorectal cancer in a subject in need thereof, comprising the step ofadministering a pharmaceutically effective dose of a material inhibitingan enzyme activity of peroxiredoxin 2 to the subject, wherein thematerial inhibiting the enzyme activity of peroxiredoxin 2 is a compoundof the following Formula 1:

wherein in Formula 1 above, R¹ is —O—R², a cyclic group, or hydrogen;and R² is a branched or unbranched C₁ to C₈ alkyl, a branched orunbranched C₂ to C₈ alkenyl, a branched or unbranched C₂ to C₈ alkynyl,or an aromatic or non-aromatic cyclic group that is substituted orunsubstituted.
 2. The method for improving or treating colorectal cancerof claim 1, wherein the material inhibiting the enzyme activity ofperoxiredoxin 2 is one or more selected from the group consisting ofCompound-1 to Compound-6 shown below:


3. The method for improving or treating colorectal cancer of claim 1,wherein the material inhibiting the enzyme activity of peroxiredoxin 2increases degradation of β-catenin.
 4. The method for improving ortreating colorectal cancer of claim 1, wherein the material inhibitingthe enzyme activity of speroxiredoxin 2 decreases degradation of Axin1by tankyrase (TNKS).
 5. The method for improving or treating colorectalcancer of claim 1, wherein the material inhibiting the enzyme activityof peroxiredoxin 2 increases oxidative inactivation of tankyrase (TNKS).6. The method for improving or treating colorectal cancer of claim 5,wherein the oxidative inactivation of tankyrase (TNKS) occurs incytoplasm of an APC-mutant cell.
 7. The method for improving or treatingcolorectal cancer of claim 1, wherein the material inhibiting the enzymeactivity of peroxiredoxin 2 decreases interaction between peroxiredoxin2 and tankyrase (TNKS) in cytoplasm of an APC-mutant cell.